Peptidoglycan cross‐linking in Staphylococcus aureus

Abstract

The peptidoglycan of Staphylococcus aureus contains relatively short glycan chains and is highly cross-linked via its peptide chains. The material from wild-type (strain H) and mutants H28, H4B and MR-1 was freed from the teichoic-acid-linked component and then hydrolysed by Chalaropsis muramidase to yield disacchariderepeating units of the glycan with attached peptides either non-cross-linked (monomer) or joined to similar units by one (dimer), two (trimer) or more (oligomer) peptide cross links. The resulting fragments were separated by high-resolution HPLC so that distinguishable components as large as nonamer could be identified. Extrapolation showed that, in S. aureus H, H28 and MR-1, oligomers at least as large as eicosamer formed part of the smooth distribution of oligomer fragments, whereas in strain H4B (PBP4-) the maximum size was around dodecamer. The oligomer distribution profile was related to the polymerization theories of Flory, which allow a distinction to be made between a monomer addition model, whereby each oligomer can only be synthesized by the addition of a single monomer unit to its next lower homologue, and a random addition model, in which an oligomer can be formed by linkage of any combination of its constituent smaller units. In S. aureus close approximation to the random addition model for oligomer synthesis and hence for peptidoglycan cross-linking was observed, both in PBP4' and PBP4-mutants. The implications for secondary cross-linking in S. aureus cell wall formation are inescapable, although the possibility of an endopeptidase/transpeptidase providing later modification of the peptidoglycan is not completely ruled out.Peptidoglycan (murein) is the major structural and stressbearing polymer of the cell walls of most prokaryotes [l]. It is composed of glycan chains of repeating N-acetylglucosaminyl-P-(I -4)-N-acetylmuramic acid (disaccharide) units substituted at the muramic acid carboxyl group with short, specific peptides [I]. The peptidoglycan network (murein sacculus [2]) is formed by the cross-linking of adjacent glycan chains via peptide linkages between the initial primary chains attached to the muramic acid. In Staphylococcus aureus the primary peptide chain has the sequence L-Ala-D-Glu(L-Lys-DAla-D-Ala) and before polymerisation of the glycan chain the &-amino group of L-lysine becomes substituted with a glycine pentapeptide [3]. Cross-links are then formed by a transpeptidase reaction, in which a terminal malanine is lost from one primary chain and the sub-terminal D-alanine becomes linked to the free amino group of a pentaglycine bridge [4]. The degree of cross-linking (CI), i.e. the number of such links achieved in practice divided by the total number possible, often expressed as a percentage (100 CI), varies greatly among bacterial species; thus in Escherichia coli and Neisseria gonorrhoeae the value is 30-40%, whereas in S. aureus it is exceptionally high at about 70-80% [4]. A value of 75% corresponds to an average degree of peptide polymerisation (Xn) of 4, since 2,...

Cell wall assembly in Staphylococcus aureus: proposed absence of secondary crosslinking reactions

“…From the results so obtained the percentage of crosslinking in the wall can be estimated (Martin & Gmeiner, 1979) at 80 YO. This figure is slightly higher than reported by Snowden & Perkins (1990) but is consistent with other estimates (Dobson & Archibald, 1978).…”

“…Thus if anionic polymer were randomly attached to muramic acid residues at different locations along the glycan chain, the larger muropeptides (containing more muramic acid residues) would have greater probabilities of being attached to anionic polymer so that differing size distributions would be seen in muropeptide fractions containing and not containing anionic polymer. The similar distribution in such fractions shown in the previous study (Snowden & Perkins, 1990) would therefore indicate a non-random location of anionic polymer. For this reason, and also because the kinetic analysis can legitimately be applied only to the total distribution, the present study has further explored the effects of fractionation procedures on muropeptide distributions.…”

“…In previous studies these negatively charged polymers have usually been removed from muramidase digests by anion-exchange chromatography (Tipper & Strominger, 1968;Oldmixon et aE., 1976;Snowden et al, 1989). It has been reported (Snowden & Perkins, 1990) that the size distribution in muropeptides associated with anionic polymer in S. aureus is similar to that in muropeptides that are not linked to anionic polymer (and thus not retained on anion-exchange resin). Comparison of attached and nonattached muropeptides is significant since it relates to the way in which anionic polymer is distributed in the cell wall.…”

“…It has not been clear whether this polymerization also proceeds by monomer addition. A recent study of crosslinking in Staphylococcus aureus (Snowden & Perkins, 1990) analysed the distribution of muropeptides formed upon digestion of cell wall with muramidase. The size distribution shown by gel-filtration HPLC was in close agreement with the distribution expected from a random polymerization mechanism.…”

Cell wall assembly in Bacillus megaterium: incorporation of new peptidoglycan by a monomer addition process

“…The distribution of muropeptides in Streptococcus faecalis (28) was also closer to that expected of a monomer addition mechanism. A similar analysis of Staphylococcus aureus gives results that are in accord with an overall random polymerization process, possibly involving an initial stage of monomer addition followed by secondary random crosslinking (31). However, it is not obvious that polymerization equations derived for homogeneous catalysis in solution can be legitimately applied to the vectorial assembly of the cell wall.…”